This invention relates to polynucleotides believed to be novel, including partial and extended sequences as well as probes and primers, constructs comprising the polynucleotides, biological materials (including plants, microorganisms and multicellular organisms) incorporating the polynucleotides, polypeptides encoded by the polynucleotides, and methods for using the polynucleotides and polypeptides. The invention relates, more particularly, to the modification of lignin content and composition in biological materials including plants, to polypeptides involved in the lignin biosynthetic pathway, and to polynucleotides encoding such enzymes.
Lignin is an insoluble polymer that is primarily responsible for the rigidity of plant stems. Specifically, lignin serves as a matrix around the polysaccharide components of some plant cell walls. The higher the lignin content, the more rigid the plant. For example, tree species synthesize large quantities of lignin, with lignin constituting between 20% to 30% of the dry weight of wood. In addition to providing rigidity, lignin aids in water transport within plants by rendering cell walls hydrophobic and water impermeable. Lignin also plays a role in disease resistance of plants by impeding the penetration and propagation of pathogenic agents.
The high concentration of lignin in trees presents a significant problem in the paper industry wherein considerable resources must be employed to separate lignin from the cellulose fiber needed for the production of paper. Methods typically employed for the removal of lignin are highly energy- and chemical-intensive, resulting in increased costs and increased levels of undesirable waste products. In the U.S. alone, about 20 million tons of lignin are removed from wood per year.
Lignin is largely responsible for the digestibility, or lack thereof, of forage crops, with small increases in plant lignin content resulting in relatively high decreases in digestibility. For example, crops with reduced lignin content provide more efficient forage for cattle, with the yield of milk and meat being higher relative to the amount of forage crop consumed. During normal plant growth, the increase in dry matter content is accompanied by a corresponding decrease in digestibility. When deciding on the optimum time to harvest forage crops, farmers must therefore chose between a high yield of less digestible material and a lower yield of more digestible material.
For some applications, an increase in lignin content is desirable since increasing the lignin content of a plant would lead to increased mechanical strength of wood, changes in its color and increased resistance to rot. Mycorrhizal species composition and abundance may also be favorably manipulated by modifying lignin content and structural composition.
As discussed in detail below, lignin is formed by polymerization of at least three different monolignols that are synthesized in a multistep pathway, each step in the pathway being catalyzed by a different enzyme. It has been shown that manipulation of the number of copies of genes encoding certain enzymes, such as cinnamyl alcohol dehydrogenase (CAD) and caffeic acid 3-O-methyltransferase (COMT) results in modification of the amount of lignin produced; see, for example, U.S. Pat. No. 5,451,514 and PCT Publication No. WO 94/23044. Furthermore, it has been shown that antisense expression of sequences encoding CAD in poplar leads to the production of lignin having a modified composition (Grand C et al., Planta (Berl.) 163:232-237, 1985).
While polynucleotides encoding some of the enzymes involved in the lignin biosynthetic pathway have been isolated for certain species of plants, genes encoding, many of the enzymes in a wide range of plant species have not yet been identified. Thus there remains a need in the art for materials useful in the modification of lignin content and composition in plants and for methods for their use.
Briefly, the present invention provides isolated polynucleotides identified in the attached Sequence Listing as SEQ ID NOS: 1-266, 350-375 and 404, variants of those sequences, constructs comprising such sequences, extended sequences comprising the sequences of SEQ ID NOS: 1-266, 350-375 and 404, and their variants, probes and primers corresponding to the sequences set out in SEQ ID NOS: 1-266, 350-375 and 404, and their variants, and polynucleotides comprising at least a specified number of contiguous residues of any of the polynucleotides identified as SEQ ID NOS: 1-266, 350-375 and 404 (x-mers), all of which are referred to herein, collectively, as xe2x80x9cpolynucleotides of the present invention.xe2x80x9d Polynucleotides of the present invention are preferably obtainable from eucalyptus and pine species, and preferably comprise open reading frames or partial open reading frames encoding enzymes, or functional portions of enzymes, involved in the lignin biosynthetic pathway. Constructs incorporating such sequences, methods for using such sequences and constructs, and biological materials, including plant cells and plants having an altered genomic and/or lignin content and composition are provided. The present invention also provides isolated polypeptide sequences identified in the attached Sequence Listing as SEQ ID NOS: 267-349, 376-401 and 405; polypeptide variants of those sequences; and polypeptides comprising the polypeptide sequences and variants of those sequences.
In one aspect, the present invention provides isolated polynucleotides encoding the following enzymes, or portions of the following enzymes: cinnamate 4-hydroxylase (C4H), coumarate 3-hydroxylase (C3H), phenolase (PNL), O-methyl transferase (OMT), cinnamyl alcohol dehydrogenase (CAD), cinnamoyl-CoA reductase (CCR), phenylalanine ammonia-lyase (PAL), 4-coumarate: CoA ligase (4CL), coniferol glucosyl transferase (CGT), coniferin beta-glucosidase (CBG), laccase (LAC), peroxidase (POX), ferulate-5-hydroxylase (F5H), alpha amylase, caffeic acid methyl transferase, caffeoyl CoA methyl transferase, coumerate 6A ligase, cytochrome P450 LXX1A, diphenol oxidase, flavonol glucosyl transferase, flavonoid hydroxylase, and isoflavone reductase.
In one embodiment, polynucleotides of the present invention encompass polynucleotides comprising a nucleotide sequence selected from the group consisting of: (a) polynucleotides recited in SEQ ID NOS: 1-266, 350-375 and 404; (b) complements of the polynucleotides recited in SEQ ID NOS: 1-266, 350-375 and 404; (c) reverse complements of the sequences recited in SEQ ID NOS: 1-266, 350-375 and 404; (d) reverse sequences of the sequences recited in SEQ ID NOS: 1-266, 350-375 and 404; and (e) variants of the polynucleotides recited in SEQ ID NOS: 1-266, 350-375 and 404. In another embodiment of the present invention, polynucleotides comprise at least a specified number of contiguous residues (x-mers) of any of the polynucleotides of SEQ ID NOS: 1-266, 350-375 and 404. In yet another aspect, polynucleotides comprise probes and primers corresponding to any of the polynucleotides of SEQ ID NOS: 1-266, 350-375 and 404.
In another aspect, the present invention provides constructs comprising a polynucleotide of the present invention, either alone or in combination with one or more of the inventive sequences, or in combination with one or more known polynucleotides; together with host cells and transgenic cells comprising such constructs.
In a related aspect, the present invention provides constructs comprising, in the 5xe2x80x2-3xe2x80x2 direction, a gene promoter sequence; an open reading frame coding for at least a functional portion of an enzyme encoded by a polynucleotide of the present invention; and a gene termination sequence. An open reading frame may be orientated in either a sense or antisense direction. DNA constructs comprising a non-coding region of a gene coding for an enzyme encoded by the above polynucleotides or a polynucleotide complementary to a non-coding region, together with a gene promoter sequence and a gene termination sequence, are also provided. Preferably, the gene promoter and termination sequences are functional in a host cell, such as a plant cell. Most preferably, the gene promoter and termination sequences are those of the original enzyme genes but others generally used in the art, such as the Cauliflower Mosaic Virus (CMV) promoter, with or without enhancers, such as the Kozak sequence or Omega enhancer, and Agrobacterium tumefaciens nopalin synthase terminator may be usefully employed in the present invention. Tissue-specific promoters may be employed in order to target expression to one or more desired tissues. In a preferred embodiment, the gene promoter sequence provides for transcription in xylem. The construct may further include a marker for the identification of transformed cells.
In a further aspect, transgenic cells, such as transgenic plant cells, comprising the constructs of the present invention are provided, together with plants comprising such transgenic cells, and fruits and seeds of such plants.
In yet another aspect, methods for modulating the lignin content and composition of a target organism such as a plant are provided, such methods including stably incorporating into the genome of the target plant a construct comprising a polynucleotide of the present invention. In a preferred embodiment, the target plant is a woody plant, preferably selected from the group consisting of eucalyptus and pine species, most preferably from the group consisting of Eucalyptus grandis and Pinus radiata. In a related aspect, a method for producing a plant having altered lignin content is provided, the method comprising transforming a plant cell with a construct comprising a polynucleotide of the present invention to provide a transgenic cell, and cultivating the transgenic cell under conditions conducive to regeneration and mature plant growth.
In yet a further aspect, the present invention provides methods for modifying the activity of an enzyme in a target organism such as a plant, comprising stably incorporating into the genome of the target organism a construct of the present invention. In a preferred embodiment, the target plant is a woody plant, preferably selected from the group consisting of eucalyptus and pine species, most preferably from the group consisting of Eucalyptus grandis and Pinus radiata. 
The present invention also provides polypeptides comprising the isolated polypeptides identified as SEQ ID NOS: 267-349, 376-401 and 405, and variants of those polypeptides.